Project Summary Transplantation is currently the treatment of choice for a number of end stage kidney diseases. Considerable progress has been made in the past decades to prevent early rejection of kidney grafts. Yet, long-term survival rates remain poor, due presumably to antibody-mediated rejection (ABMR). Although antibodies specific to donor HLA molecules (DSA) indisputably contribute to mechanisms of ABMR, other types of antibodies, especially autoantibodies, have also been implicated. Previous studies from our lab associated natural antibodies (Nabs) reactive to apoptotic cells with ABMR. Moreover, we found that high level of IgG Nabs in pre-transplant serum correlated with long-term graft loss independently of anti-HLA antibodies. Based on this data, we hypothesize that Nabs are important elements in the development of ABMR and subsequently graft loss. Here, we propose to continue these studies by extending our analysis to a larger group of patients treated at other centers. We will also investigate the source of Nabs and characterize the pathogenic potential of these antibodies in vivo. Our experiments will be carried out in 3 aims: Aim-1. To determine whether serum IgG Nabs correlate with ABMR and long-term kidney graft loss. We will carry out a blinded multi-center large-scale assessment of IgG Nabs in pre- and post-transplant serum samples from an extensive cohort of kidney transplant recipients. Serum IgG Nabs measurements will be correlated with donor specific antibodies, other non-HLA antibodies as well as a comprehensive series of clinical parameters including different types of rejection and causes of graft loss. A predictive model will then be built to evaluate the clinical relevance of Nabs levels as a significant factor predicting rejection and graft failure. The synergistic effect between Nabs, DSA and other types of antibodies will also be evaluated. Aim 2. To determine the source of Nabs during ABMR. Using ELISPOT assays we will assess the frequency of IgM and IgG Nabs-producing cells in the blood and graft tissue of kidney transplant recipients and determine whether this frequency correlates with serum Nabs levels and ABMR. We will also attempt to characterize the phenotype of Nabs-producing innate B cells. Aim 3. To verify the pathogenicity of Nabs in vivo. In this aim we will use a mouse aortic transplantation model to study the pathogenicity of IgG Nabs in vivo. Through passive transfer of Nabs and DSA in transplanted mice, we will also investigate the synergistic effect of both types of antibodies. Lastly, we will use a series of mutant mice to investigate the molecules, immune cells and pathways involved in Nabs-mediated vascular rejection.